Table 1.
Antibodies utilized for oligodendrocyte lineage cell characterization by flow cytometry.
Figure 1.
Flow cytometry for oligodendroglial analysis.
Flow cytometry allows for rapid analysis of eight or more proteins expressed on individual cells. Cells are streamed through a narrow flow cell sequentially and interrogated individually, thus largely static organs such as the brain must be dissociated into individual cells prior to analysis. Cells are stained with a cocktail of antibodies, each conjugated to a unique fluorochrome. Because of distinct proteins changes that occur during the maturation of the oligodendrocyte, antibodies can be utilized to characterize cells throughout the oligodendrocyte lineage. Thousands of cells per second are streamed through the flow cytometer, excited by laser light, and emitted fluorescence is directed through filters to photomultiplier tubes specific for each fluorochrome. For subsequent analysis, single cells are distinguished from small debris using forward (FSC) and side (SSC) scatter of light. Live cells are distinguished from dead cells using a live/dead dye, and CNS resident cells are distinguished from residual hematopoietic cells using a CD45 antibody. The entire oligodendrocyte lineage, from early progenitor to mature oligodendrocyte, can be analyzed and quantified.
Figure 2.
Optimization of oligodendroglial isolation and purification for flow cytometry.
(A) Brains were removed from C57BL/6 mice, coarsely chopped, and dissociated with 1 ml of one of eight dissociation enzyme for 30 min at 37°C: Accutase, Liberase DL, Liberase DH, Liberase TL, Liberase TM, Liberase TH, TrypLE, 0.25% Trypsin-EDTA, or PBS. Digested tissues were filtered, and single cells were purified using density centrifugation. Total cells were calculated by haemocytometer. Live cells and dead cells were stained with fluorescent dyes, and the ratio of live/dead cells was analyzed by flow cytometry. (B) Brains were removed from mice and dissociated with 1 ml of one of five dissociation enzymes for 30 min at 37°C. Single cells were purified by density centrifugation and stained with anti-A2B5, -O4, and -O1 antibodies for 30 min on ice. Positive cellular staining was quantified by flow cytometry. (C) Single cell suspensions were purified from Accutase-dissociated CNS tissue by density centrifugation or bead-based myelin removal technology. Representative forward (x-axis) and side (y-axis) scatter of light plots are illustrated for each fraction following purification. (D) Total numbers of cells in each fraction was determined by haemocytometer. (E–G) Single cell fractions, following density centrifugation (E–G) or myelin removal bead (F) purification of CNS tissue, were stained with anti-A2B5, -PDGFRα, -NG2, -O4, -CC1, or O1 for 30 min on ice. Percentages of positive cellular staining were quantified by flow cytometry and absolute cell numbers were back calculated from total cell counts. Data are representative of three independent experiments
Figure 3.
Oligodendrocyte lineage cell antibody titrations.
Brains were removed from C57BL/6 mice, coarsely chopped, and dissociated with 1 ml of Accutase for 30 min at 37°C. Digested tissues were filtered, and single cells were purified using density centrifugation. Cells were stained with 0.5 µg, 1 µg, or 5 µg of each antibody or isotype control antibody for 30 min on ice. Positive cellular staining was quantified by flow cytometry.
Figure 4.
Oligodendrocyte lineage analysis by flow cytometry using cultured oligodendrocyte progenitor cells (OPCs) and differentiated oligodendrocytes.
Primary OPCs were isolated from C57BL/6 postnatal day five pups by O4 immunopanning and grown for six days in the presence of or five days in the absence of the mitogen PDGF. (A) Representative photomicrographs of cultures. (B) Cultured OPCs and oligodendrocytes were lifted from plates, stained with antibodies against A2B5, O4, O1, MOSP, and GALC, and analyzed by flow cytometry. (C) Double labeling analysis. Gates were drawn for double positive populations: A2B5+O4+ intermediate OPCs, O4+O1+ late OPCs, O1+GALC+ and GALC+MOSP+ mature oligodendrocytes. (D) Adult C57BL/6 CNS cells were isolated, stained with oligodendroglial antibodies, and sorted by FACS into three populations: A2B5+PDGFRα+ early OPCs, NG2+O4+ intermediate OPCs, GALC+MOG+ mature oligodendrocytes. Representative sorting flow plots are shown. mRNA was purified and transcription levels of stage-specific oligodendroglial genes Olig2, Pdgfra, Cspg4, Plp1, Mbp, and Mog were quantified by RT-PCR. Data were normalized to a housekeeping gene and are reported relative to the A2B5+PDGFRα+ sorted population. Error bars indicate SD. *P≤0.05; **P≤0.01; ***P≤0.001 for post-hoc analysis. Data are representative of three independent experiments.
Figure 5.
Oligodendroglial lineage analysis during development.
Postnatal day four through ten C57BL/6 mice were sacrificed, brains extracted, and CNS tissue prepared for flow cytometry (n≥3/time point). Cells were stained with an antibody panel consisting of anti-A2B5, -NG2, -GALC, and -CD45 and analyzed by flow cytometry to characterize changes in oligodendroglial populations. (A) To examine CNS resident cells, single cells were gated, followed by live cells, followed by CD45− gate. The GALC+ mature oligodendrocyte population was defined, and the A2B5+ and NG2+ oligodendrocyte progenitor cell (OPC) populations were defined from the GALC− gate. (b) Representative oligodendroglial analysis from postnatal day 7 and 10 mice. Upper histograms illustrate GALC+ mature oligodendrocyte staining. Lower plots indicate A2B5+NG2− early OPC and A2B5+NG2+ intermediate OPC populations. (C) Total cell yields from brains of individual mice, day 4 vs. day 10, P = 0.0013. (D) A2B5+NG2−GALC− early OPCs (circles), A2B5+NG2+GALC− intermediate OPCs (squares), and A2B5−NG2−GALC+ mature oligodendrocytes (triangles) are depicted. Error bars represent SD of mice per time point. Early OPCs: day 4 vs. 7, P≤0.001; day 7 vs. 10, P≤0.01. Intermediate OPCs: day 7 vs. 8, P≤0.001; day 8 vs. 10, P≤0.001. Mature oligodendrocytes: day 8 vs. 10, P≤0.05. Data are representative of three independent experiments.
Figure 6.
Characterizing oligodendroglial population changes through cuprizone-induced demyelination.
C57BL/6 mice were fed 0.2% cuprizone chow for five weeks and returned to a normal diet for a sixth week. A cohort of mice was sacrificed weekly throughout the disease course, brains were extracted, and CNS processed for flow cytometry (n = 5). Naïve mice were concurrently assayed at each time point for standardization (n = 3). (A) Cells were first gated by forward and side scatter then single cells were gated. Live cells were next gated, and hematopoietic cells excluded by gating on CD45-/low cells. (B) From the CNS resident cells, oligodendroglial populations were defined: A2B5+PDGFRα+ early OPCs, PDGFRα+O4+ intermediate OPCs, O4+O1+ pre-myelinating oligodendrocytes, and O1+GALC+ mature oligodendrocytes. Mature oligodendrocytes (C), pre-myelinating oligodendrocytes (D), early OPCs (E), and late OPCs (F) were compared to naïve mice at each time point. CD45high peripheral immune cells (G) and CD45low microglia (H) were also quantified. Error bars indicate SD of mice per time point. *P≤0.05; **P≤0.01; ***P≤0.001 for post-hoc analysis. Data are representative of two independent experiments.
Figure 7.
Characterizing oligodendroglial populations through relapsing-remitting EAE.
SJL/J mice were immunized with PLP139–151 and scored daily for clinical disease. A cohort of mice was sacrificed at disease onset, peak, remission, and relapse, and spinal cords were analyzed by flow cytometry (n = 5). Naïve mice were also assayed at each time point for standardization across days (n = 3). (A) Cells were distinguished from debris by forward and side scatter then singlet cells were gated. Live cells were gated by dead cell exclusion, and CNS resident cells were identified as CD45− or CD45low. (B) Oligodendroglial cells were defined by double positive staining: A2B5+PDGFRα+ early OPCs, A2B5+NG2+ intermediate OPCs, NG2+O4+ late OPCs, O4+MOG+ pre-myelinating oligodendrocytes, and GALC+MOG+ mature oligodendrocytes. (C) The complete oligodendroglial lineage is illustrated for the naïve spinal cord. Oligodendroglial populations at each stage of disease were compared to naïve spinal cord. The table indicates number and percentage of CD45-/low CNS resident cells ± SD per group of mice. (D) Clinical relapsing-remitting disease course and sacrifice points. (E,F) CD45high infiltrating immune cells and CD45low microglia in the spinal cord were also quantified throughout EAE disease. *P≤0.05; **P≤0.01; ***P≤0.001 for post-hoc analysis. Data are representative of three independent experiments.